Production and characterization of transgenic mice systemically expressing endo-β-galactosidase C

The alpha Gal epitope (Gal alpha 1-3Gal) is a sugar structure expressed on the cell surface of almost all organisms except humans and old-world-monkeys, which express natural anti-alpha Gal antibodies. The presence of these antibodies elicits a hyper acute rejection (HAR) upon xenotransplantation of cellular materials, such as from pigs to human beings. Endo-beta-galactosidase C (EndoGalC), an enzyme isolated from Clostridium perfringens, removes the alpha Gal epitope by cleaving the Gal beta 1-4GlcNAc linkage in the Gal alpha 1-3Gal beta 1-4GlcNAc sequence. To explore the possibility that cells or organs from transgenic pigs systemically expressing EndoGalC might be suitable for xenotransplantation, we first introduced the EndoGalC transgene into the mouse genome via pronuclear injection. The progeny of the resulting transgenics expressed EndoGalC mRNA and protein. Flow cytometry and histochemical analyses revealed a dramatic reduction in the expression of the alpha Gal epitope in these mice. They also exhibited abnormal phenotypes, such as occasional death immediately after birth, growth retardation, and transient skin lesions. Interestingly, the phenotypic abnormalities seen in these transgenics were similar to those observed in beta 1,4-galactosyltransferase 1 (beta 4GalT-1) knockout (KO) mice. Most probably, these phenotypes were caused by exposure of the internal N-acetylglucosamine residue at the end of the sugar chain on the cell surface. The present findings also provide some basis for evaluating possible application of the transgenic approach for xenotranplantation.